Mining for Active Molecules in Probiotic Supernatant by Combining Non-Targeted Metabolomics and Immunoregulation Testing

Chronic respiratory diseases such as asthma are highly prevalent in industrialized countries. As cases are expected to rise, there is a growing demand for alternative therapies. Our recent research on the potential benefits of probiotics suggests that they could prevent and reduce the symptoms of many diseases by modulating the host immune system with secreted metabolites. This article presents the first steps of the research that led us to identify the immunoregulatory bioactivity of the amino acid d-Trp reported in our previous study. Here we analyzed the cell culture metabolic footprinting of 25 commercially available probiotic strains to associate metabolic pathway activity information with their respective immune modulatory activity observed in vitro. Crude probiotic supernatant samples were processed in three different ways prior to untargeted analysis in positive and negative ionization mode by direct infusion ESI-FT-ICR-MS: protein precipitation and solid phase extraction (SPE) using HLB and CN-E sorbent cartridges. The data obtained were submitted to multivariate statistical analyses to distinguish supernatant samples into the bioactive and non-bioactive group. Pathway analysis using discriminant molecular features showed an overrepresentation of the tryptophan metabolic pathway for the bioactive supernatant class, suggesting that molecules taking part in that pathway may be involved in the immunomodulatory activity observed in vitro. This work showcases the potential of metabolomics to drive product development and novel bioactive compound discovery out of complex biological samples in a top-down manner.